Assay based on doped nanoparticles

ABSTRACT

The invention relates to an assay based on resonance energy transfer (RET), comprising a first molecule grope A, which is marked with at least one energy donor, and at least one second molecule group B which is marked with at least one energy acceptor, the donor comprising a molecule or particle which can be energetically excited by an external radiation source and which is fluorescence enabled and the acceptor comprising a molecule or particle which can be excited by energy transfer via the donor with partial or complete quenching of the donor fluorescence, and the donor and/or acceptor comprise luminescing inorganic dope nanoparticles having an expansion of ≦50 nanometers, emitting electromagnetic radiation with stokes or anti-stokes scattering after energetic excitation.

This application is a 371 of PCT/EP02/12256, filed Nov. 4, 2002, andclaims priority under 35 USC §119 on the basis of German Application No.101 53 829.4, filed Nov. 5, 2001.

The present invention relates to a biotechnological assay which is basedon a resonance energy transfer (RET) and which can be used to detectbiological molecules such as enzymes, antibodies, nucleic acid-bindingmolecules, nucleic acids, polynucleotides or morpholino.

Immunoassays or nucleic acid detection methods are the basis of manyapplications in medical diagnosis, in the production control ofbiotechnologically produced products and in research. One method, whichis used here, is that of resonance energy transfer (RET) between dyes.

The principle of resonance energy transfer (RET) is based onradiationless transfer of energy from a donor which is capable offluorescing to an acceptor which is located in spatial proximity. Thistechnique can be used to determine distances at the molecular level in arange between approx. 1 and 8 nm. The energy which has been transferredto the acceptor can, on the one hand, relax in a radiationless manner bymeans of internal conversion (RET) and then only leads to the donorfluorescence being quenched. On the other hand, the transferred energycan also be emitted by means of the acceptor fluorescing. This isreferred to as fluorescence resonance energy transfer (FRET). Thesephenomena have been well understood for a long time now and, in the caseof a dipole-dipole interaction between donor and acceptor, explained byFörster's theory (see, e.g., J. R. Lakowicz, “Principles of FluorescenceSpectroscopy”, Kluwer Academic Press, New York, 1999, pages 368-445).The energy transfer reduces the intensity of the donor fluorescence andits decay time and correspondingly increases the fluorescence of theacceptor or else only excites or sensitizes it for the first time. Theefficiency E of the energy transfer is very sensitive to the distance Rbetween the donor and acceptor and declines proportionally to R₀ ⁶/(R₀⁶+R⁶). The mean range of the energy transfer, at which the efficiency is50%, is defined by means of a material-specific constant, i.e. theFörster radius R₀, and lies in the range of a few nanometers (less than10 nm). When the excited state of the acceptor relaxes in aradiationless manner it is only donor fluorescence which suffersanything from a reduction through to complete quenching. In that whichfollows, the term (F)RET is used when the terms RET and FRET can be usedalternately. (F)RET-capable donor/acceptor pairs are termed (F)RETpairs. In that which follows, the terms fluorescence and luminescenceare used synonymously.

In biological systems, (FRET) is used to detect the spatial proximity toeach other of appropriately labeled biomolecules or molecular groups.This method can be used as a method for detecting protein-proteininteractions, e.g. as a method for detecting the antigen-antibodyreaction in immune reactions, a receptor-ligand interaction, a nucleicacid hybridization or the binding of proteins to nucleic acids. Thedetection is itself effected by means of measuring the change in theintensity of, or the spectral change in, the donor fluorescence oracceptor fluorescence or by means of measuring a change in the decaytime of the donor fluorescence. A large number of applications in thisregard are described in the literature, such as the detection ofspecific antigens in immunofluorescence assays (U.S. Pat. Nos.3,996,345; 4,160,016; 4,174,384; 4,199,559), the determination of theelectrostatic potential in specific, localized regions on the surface ofproteins (Yamamoto et al.; J. Mol. Biol. 241, 1994, pages 714-731), orthe method involving high-throughput screening (Boisclair et al.; J. ofBiomolecular Screening, 5, 2000, pages 319-328).

(F)RET systems are also used to determine absolute distances between twomolecules or within a biomolecule. Two labels, which measurably interactin dependence on their distance from each other, are introduced for thispurpose. Known applications of this method are the analysis of proteinstructure or DNA structure (Heyduk et al.; SPIE Vol. 3256, 1998, pages218-222), the determination of distances within polypeptides (Lakowiczet al.; Biophys. Chem. 36, 1990, pages 99-115), proteins (K. Cai et al.;J. Biol. Chem. 271 1996, pages 27311-27320), polynucleotides(Hochstrasser et al.; Biophys. Chem. 45, 1992, pages 133-141 and Ozakiet al.; Nucl. Acids Res. 20, 1992, pages 5205-5214) or othermacromolecules, the investigation of membranes and membrane proteins andtheir structure (S. Wang et al.; Biochemistry 27, 1988, pages2033-2039), and the detection (U.S. Pat. Nos. 4,996,143; 5,532,129;5,565,332) and quantification of nucleic acids which have been amplifiedby PCR (polymerase chain reaction) (U.S. Pat. Nos. 5,538,848;5,723,591), e.g. for in vitro diagnosis, genetic analysis, forensics,foodstuff tests, agricultural product tests or parenthood tests. DNA orRNA is detected or quantified directly, i.e. without any additionalseparation steps.

The 5′-nuclease assay (U.S. Pat. Nos. 5,538,848; 5,210,015; Holland etal.; Proc. Natl. Acad. Sci USA 88, 1991, pages 7276-7280; Lee et al.;Nucleic Acids Res. 21, 1993, pages 3761-3766) which is termed theTaqMan® assay (Applied Biosystems Division of Perkin-Elmer Corp., FosterCity, USA) is a quantitative nucleic acid determination by means ofreal-time PCR which uses (F)RET systems. The method of molecular beacons(Tyagi and Kramer, Nature Biotechnology 14, 1996, pages 303-306; U.S.Pat. No. 5,312,728) is based on a similar mechanism.

Organic dye molecules such as fluorescein, cyanine or rhodamine, forexample, are classical, commercially available materials for makingefficient (F)RET pairs. A general disadvantage of these organicfluorescent dyes is that they frequently exhibit a stability towardincident light which is inadequate for many applications. Particularlyin the presence of oxygen or free radicals, some of them can already beirreversibly damaged or destroyed after a few million lightabsorption/light emission cycles. Furthermore, the fluorescent organicdye molecules can also have a phototoxic effect on biological materialin the vicinity. On the one hand, the broad emission bands of theorganic fluorescent dyes, with their additional ramifications into thelong-wave region of the spectrum, are unfavorable for simultaneouslyreading several dyes, i.e. what is termed multiplexing. On the otherhand, their usually small Stokes shift, i.e. the difference between theexcitation maximum and the emission maximum of a dye and the relativelynarrow spectral excitation bands within which an excitation is possible,is disadvantageous. As a result, several light sources and/or elaboratefilter systems frequently have to be used, thereby additionallyrestricting the simultaneous reading of several dyes.

It is also possible to use fluorescent proteins as a FRET pair. In thiscase, the FRET process involved is also termed a bioluminescenceresonance energy transfer (BRET). The fluorescent proteins include thegreen fluorescent protein GFP (U.S. Pat. No. 5,491,084) and its variantswhich possess other absorption and/or emission maxima, such as theyellow (YFP) or cyan (CFP) fluorescent proteins (U.S. Pat. No.5,625,048). In this connection, GFPs can either be used as the donor andacceptor or in combination with other fluorophores such as fluoresceinor luciferase (review article: Pollok and Heim; Trends Cell Biol. 9,1999, pages 57-60). A problem is the small selection of different GFPproteins which satisfy the requirements for a suitable FRET pair(sufficiently large difference in the excitation wavelengths, sufficientoverlapping of the donor and acceptor emission and excitationwavelengths). Thus, it has to date only been possible to successfullyapply two combinations of GFPs as a FRET pair (review article: Pollokand Heim; Trends Cell Biol. 9, 1999, pages 57-60). Even in combinationwith other dyes or bioluminescent proteins, the small number andmarkedly different intensities of the GFPs is a limiting factor.

As an alternative to organic dyes, metal chelates or metal complexes arealso used for FRET (see, e.g., Selvin; IEEE J. of Selected Topics inQuantum Electronics 2, 1996, pages 1077-1087).

Lanthanide chelates can be employed either as (F)RET pairs (Clark etal., Anal. Biochem. 210, 1993, pages 1 ff.) or as only the donor whichtransfers the energy to organic fluorescent dyes (Thomas et al.; Proc.Natl. Acad. Sci. 75, 1978, pages 5746 ff; S. G. Jones et al.; Journal ofFluorescence 11, 2001, pages 13-21) or to quenchers (Tanaka et al.; J.Photochem. Photobiol. A 74, 1993, pages 15 ff; Marko et al.;Biochemistry 31, 1992, pages 703 ff.).

Systems or assays which are based on energy transfer and onfluorochromes and chelates having a long lifetime have been disclosed ina number of patents (WO 8 707 955, EP 242 527, EP 439 036, WO 9201225,U.S. Pat. Nos. 4,822,733, 5,279,943, 5,622,821, 5,656,433, 5,998,146,6,239,271). They use time gated fluorimetry (TGF) and/or time-resolvedfluorimetry (TRF) for detecting an analyte.

In this connection, TGF is understood as being a measurement mode inwhich the excitation is effected using a pulsed light source (laser,photoflash lamp) and, after a defined delay time which then follows, thelight emission is measured within a given time window. The delay time issufficiently long to detect the long-lived fluorescence of thelanthanide chelates with an adequately high intensity. However, thedelay time virtually complete discriminates against the short-livedbackground fluorescence (usually <1 μs) which is elicited by intrinsicautofluorescence of the biological material, impurities in the solventor surrounding vessel materials. In contrast to the TGF mode,measurements carried out in the TRF mode measure the fluorescence as afunction of time at a fixed wavelength. In this connection, the donor isalso excited by a pulsed light source or else by a light source whichhas been modulated in some other way.

However, a disadvantage of the lanthanide chelates or metal complexes isthe fact that their chemical stability is low for a number ofapplications or that their fluorescence properties depend on thechemical environment of the particles. Frequently, additional separationsteps, or an additional complex formation, is/are also required in orderto be able to measure a fluorescence.

FRET effects have also been observed in the case of particulate labelsystems which are based on semiconductor nanocrystals, what are termedquantum dots: (Bawendi et al. and C. Kagan et al.; Phys. Rev. Lett. 76,1996, pages 1517-1520). Quantum dots are also able to interact withorganic fluorophores (O. Schmelz et al.; Langmuir 17, 2001, pages2861-2865).

It is possible to exploit FRET effects between quantum dots themselvesor else between quantum dots and other substances (e.g. dyes). WO00/29617 discloses that it is possible to detect proteins and nucleicacids using quantum dots as labels. In particular, the patent alsodiscloses their use as fluorophores in the case of the hairpin-like DNAstructures known as “molecular beacons”.

However, a disadvantage of the quantum dots is that they have to beproduced with the highest possible degree of precision. Since theemission wavelength of the fluorescent light depends on the size of thequantum dots, it is necessary to achieve a very narrow particle sizedistribution in a sample. In order to ensure that the fluorescent lightis of the narrow band width which is required for the multiplexing, thedifferences in size between quantum dots of one species can only be afew Angströms, i.e. amount to only a few monolayers. This places highdemands on the synthesis. In addition, due to radiationlesselectron/hole pair recombinations on their surface, quantum dotsnormally exhibit relatively weak quantum efficiencies. For this reason,it is necessary to produce core-shell structures (Xiaogang Pent et al.;J. Am. Chem. Soc. 119, 1997, pages 7019-7029), which require a moreelaborate synthesis, in order to increase the quantum efficiencies.

Furthermore, in the case of quantum dots, the decay time of thefluorescence is very short and is in the lower nanosecond range. Forthis reason, it is not possible to make any measurements in the TGF modeand only possible to make measurements in the TRF mode using relativelyelaborate technology and equipment. Another disadvantage of the quantumdot systems is their composition, with many of the systems containingtoxic elements such as cadmium, selenium or arsenic.

Nano-scale phosphors which are of less than 50 nm in size, and which aredesignated luminescent inorganic doped nanoparticles (lid nanoparticles)below, have been described many times in scientific publications.

The published lid nanoparticles consist of oxides, sulfides, phosphatesor vanadates which are doped with lanthanides or with Mn, Al, Ag or Cu.These lid nanoparticles fluoresce, due to their doping, in a narrowspectral range. The preparation of the following lid nanoparticles hasbeen published, inter alia: LaPO₄:Ce,Th; (K. Riwotzki et al.; AngewandteChemie, Int. Ed. 40, 2001, pages 573-576); YVO₄:Eu, YVO₄:Sm, YVO₄:Dy (K.Riwotzki, M. Haase; Journal of Physical Chemistry B; Vol. 102, 1998,pages 10129-10135); LaPO₄:Eu, LaPO₄:Ce, LaPO₄:Ce,Tb; (H. Meyssamy, K.Riwotzki, A. Kornowski, S. Naused, M. Haase; Advanced Materials, Vol.11, Issue 10, 1999, pages 840-844); (K. Riwotzki, H. Meyssamy, A.Kornowski, M. Haase; Journal of Physical Chemistry B Vol. 104, 2000,pages 2824-2828); ZnS:Tb, ZnS:TbF₃, ZnS:Eu, ZnS:EuF₃, (M. Ihara, T.Igarashi, T. Kusunoki, K. Ohno; Society for Information Display,Proceedings 1999, Session 49.3); Y₂O₃:Eu (Q. Li, L. Gao, D. S. Yan;Nanostructured Materials Vol. 8, 1999, pages 825 ff); Y₂SiO₅:Eu (M. Yin,W. Zhang, S. Xia, J. C. Krupa; Journal of Luminescence, Vol. 68, 1996,pages 335 ff.); SiO₂:Dy, SiO₂:Al, (Y. H. Li, C. M. Mo, L. D. Zhang, R.C. Liu, Y. S. Liu; Nanostructured Materials Vol. 11, Issue 3, 1999,pages 307-310); Y₂O₃:Tb (Y. L. Soo, S. W. Huang, Z. H. Ming, Y. H. Kao,G. C. Smith, E. Goldburt, R. Hodel, B. Kulkarni, J. V. D. Veliadis, R.H. Bhargava; Journal of Applied Physics Vol. 83, Issue 10, 1998, pages5404-5409); CdS:Mn (R. N. Bhargava, D. Gallagher, X. Hong, A. Nurmikko;Physical Review Letters Vol. 72, 1994, pages 416-419); ZnS:Tb (R. H.Bhargava, D. Gallagher, T. Welker; Journal of Luminescence, Vol. 60,1994, pages 275 ff.). Ullmann's Encyclopedia of Industrial Chemistry,WILEY-VCH, 6^(th) edition, 2001 Electronic Release, The “LuminescentMaterials: 1. Inorganic Phosphors” chapter, provides a review of theknown luminescent inorganic doped materials which are of a fewmicrometers in size, and of their use as industrial phosphors.

In many cases, a (F)RET between a donor (sensitizer) and an acceptor(emitter) is also responsible for the light emission which is elicitedin luminous phosphors as are used, for example, for fluorescent lamps(D. Dexter; J. Chem. Phys. 21, 1953, pages 836-850, T. Jüstel et al.;Angewandte Chemie, International Edition 37, 1998, pages: 3084-3103).Since, however, donor and acceptor are present in a shared crystallattice in the case of a luminescent phosphor, the (F)RET system of theluminescent phosphors cannot be used for detecting a parameter changeresulting from a biochemical process.

U.S. Pat. No. 5,043,265 discloses that it is possible to detectbiological macromolecules which are coupled to nanoscale luminescentphosphor particles by measuring the fluorescence. The patent explainsthat the fluorescence of the particles will rapidly lose its intensityas the diameter becomes smaller and that the particles should thereforebe larger than 20 nm and preferably even larger than 100 nm.

U.S. Pat. No. 5,674,698 discloses special types of luminous phosphorsfor use as biological labels. These luminous phosphors are “upconvertingphosphors” which have the property of emitting light, whose wavelengthis shorter than that of the absorbed light, by way of a two-photonprocess. Using these particles makes it possible to work almost free ofbackground since such autofluorescence is to a very large extentsuppressed. The particles are prepared by milling and then annealing.The particle size is between 10 nm and 3 μm, preferably between 300 nmand 1 μm. These particles are primarily used in immunoassays (see, forexample, Niedbala et al.; Analytical Biochemistry 293, 2001, pages22-30). One disadvantage of these particles is their broad sizedistribution resulting from the preparation process. Another is that, inthe case of the smaller particles, there are frequently qualitativerestraints which result from the preparation and which are reflected inthe preferred particle size of 300 nm-1 μm. In general, a higherexcitation intensity than in the case of the one-photon process isrequired for exciting a two-photon process in order to achievecomparable emission intensity.

U.S. Pat. No. 6,159,686 discloses special phosphor/dye complexes forcarrying out photophysical catalysis or photodynamic therapy.Upconverting phosphors are initially excited with innocuous infraredlight. Energy in the visible light range is then transferred to the dye,which in turn, acting as a catalyst, transfers its energy to a targetmolecule. It is also possible to detect target analytes using such pairsof upconverting phosphors and suitable dyes. For this, a complexcomposed of target analyte, phosphor and dye is formed in the presenceof the target analyte, with this complex then permitting, as a result ofthe spatial proximity, an energy transfer from the phosphor to the dye.This patent also discloses the use of these upconverting phosphors,together with a corresponding “matched label” in homogeneous,heterogeneous and competitive assays. In this connection, the phosphorscan be used either as donor or as acceptor.

The object according to the invention consists in providing an assay fordetecting a biological target molecule, which assay does not suffer fromthe disadvantages described in the prior art.

The object is achieved, according to the invention, by means of an assaywhich is based on resonance energy transfer (RET) or on fluorescenceresonance energy transfer (FRET) and which contains a first moleculegroup A, which is labeled with at least one energy donor according tothe invention, and at least one second molecule group B, which is ineach case labeled with at least one energy acceptor.

Within the meaning of the invention, a donor is understood as being amolecule or particle which is energetically excited, continuously or ina time-modulated manner, by an external radiation source(electromagnetic radiation or particle radiation) and which is capableof fluorescence.

Within the meaning of the invention, an acceptor is understood as beinga molecule or particle which is excited by energy transfer by way of thedonor, which completely or partly quenches donor fluorescence and whichcan, but which does not have to, itself be capable of fluorescence. Adonor which is not capable of fluorescence relaxes in a radiationlessmanner.

According to the invention, donor and/or acceptor comprise lidnanoparticles which have a breadth of ≦50 nanometers and which, after anenergetic excitation, emit electromagnetic radiation with a Stokes orAntistokes shift.

The advantage of an assay which is based on lid nanoparticles having abreadth of 50 nanometers or less is that the particles exhibit lesspotential for steric problems or undesirable sedimentation in an assaythan can be the case when using larger particles. In addition, thepresence of the lid nanoparticle has less influence on the kinetics of abinding reaction (e.g. immune reaction or DNA hybridization) or of abiochemical process which is to be investigated.

In this, larger lid particles suffer from disadvantages when makingmeasurements in the TRF mode. In the case of a (F)RET, it is onlypossible for energy to be transferred from, for example, a lid particle,which is acting as donor, to a molecule which is located in spatialproximity, and which is acting as acceptor, within a distance extendinga few nanometers. This means that, in the case of relatively largeparticles, a significant part of the particle volume, and consequentlyof the doping ions in the particle, is not within range of the acceptorwhich is located in front of the particle surface and is therefore notinvolved in the (F)RET. As a result, the effect of the decay time change(TRF mode) caused by a (F)RET is less pronounced or possibly no longermeasurable. For the same reasons, a complete, or at least significant,quenching of the donor fluorescence by the acceptor would no longer bepossible.

A RET or FRET can be effected by means of a dipole/dipole interaction(Förster transfer), by means of an interaction with involvement ofhigher multipoles, or by means of the migration of charges or excitons.In the case of a Förster transfer, the spectral overlap between thedonor emission and the acceptor absorption must be sufficiently large.The distance between the donor and acceptor can consequently be measuredsince the efficiency of the energy transfer depends on the distance.

Preference is given to at least one of the two partners (donor oracceptor) being a luminescent inorganic doped nanoparticle having a longfluorescence decay time (>5 ns). The other partner in each case eithercontains a molecular, organic chromophore or a luminescent inorganicdoped nanoparticle which preferably exhibits a shorter fluorescencedecay time. In this connection, the lid nanoparticle having a longfluorescence decay time has a halflife of more than 5 ns, preferablybetween 1 μs and 50 ms, and particularly preferably between 100 μs and10 ms. In an assay of this design, the donor can be excited with apulsed light source of suitable wavelength. When the donor/acceptor pairare in appropriate spatial proximity to each other (a few nanometers), aFRET can now take place, i.e. the acceptor, e.g. a molecularchromophore, is sensitized and can release its energy by means of lightemission. Since the decay time e.g. of the donor fluorescence is verylong, the decay time of the sensitized fluorescence of the acceptor isalso very long, on account of the FRET, and consequently very muchlonger than as a result of the direct excitation of the acceptor by thepulsed light source. When the fluorescence is measured in the TGF mode,it is therefore possible, by masking out the short-lived acceptorfluorescence, to detect the sensitized acceptor fluorescence virtuallyin the absence of background and consequently with a high degree ofsensitivity. By using a light source which is modulated with a suitablefrequency, it is also possible to carry out phase-sensitivemeasurements. The donor can also exhibit short-lived fluorescence andthe acceptor exhibit long-lived fluorescence, as can be observed, forexample, with the system of doped LaPO₄ nanoparticles. In this case,those nanoparticles which are doped with Cer ions act as donor and thosewhich are doped with terbium ions act as acceptor.

In another embodiment, the donor consists of a lid nanoparticle and theacceptor consists of a conducting material. These materials can bemetals, such as gold (Au), silver (Ag) or platinum (Pt), or conductingoxides, such as indium tin oxide (ITO), or conducting polymers. In thisconnection, they can be present in particulate form as nanoparticles ormicroparticles, or consist of a planar surface, which can also bestructured.

The lid nanoparticles which are used in the assay according to theinvention are doped with foreign ions such that the can be excited bynarrow-band or broad-band, pulsed, modulated or continuouselectromagnetic radiation with wavelengths in the range of infraredlight, of visible light, of UV, of X-ray light or of γ-radiation orparticle radiation, such as electron radiation, or by a particle beam,and the acceptor can be qualitatively and/or quantitatively detected bytime-resolved or continuous measurement of material-specific fluorescentlight or its change.

The biochemical reaction is detected by measuring a RET or FRET, i.e. bymeasuring the change in the luminescence properties (intensity, spectralor by a change in the decay time) of the lid nanoparticles and/or theother chromophores involved. In this way, it is possible to detect, inan assay, the changes in the spacing of the (F)RET partners involved.

The spatial proximity of an acceptor can be detected, in a (F)RETsystem, from the change in the donor decay time. Because of the presenceof another decay channel, due to the transfer of energy to the acceptor,the decay time of the donor fluorescence is significantly shortened.This change can be measured both in the case of the donor fluorescenceand in the case of the sensitized acceptor fluorescence (measurement inthe TRF mode). The emission decay time, as an observed quantity forFRET, offers an alternative to measuring intensities. It makes ameasurement which is independent of concentration effects, quantumefficiency of the chromophore, incomplete labeling and partial orcomplete quenching of the acceptor fluorescence. Virtually every photonwhich is detected is a contribution to the useful signal. In the case ofa Förster transfer, and when using the mathematical relationships whichare known for this purpose, it is also possible to infer the spatialdistance between the donor and the acceptor from the decrease in thedecay time of the donor fluorescence. An important advantage of usinglid nanoparticles is their intrinsically long decay time, whichfrequently extends into the range of a few milliseconds and cantherefore be conveniently recorded using simple experimental means.

The lid nanoparticles have a virtually spherical morphology, withbreadths in the range from 1 nm to 50 nm, preferably in the range from 1nm to less than 20 μm, and particularly preferably in the range from 2nm to 10 nm. Breadths are understood as meaning the maximum separationof two points lying on the surface of a lid nanoparticle. The lidnanoparticles can also have an ellipsoidal morphology or be faceted,with breadths which lie within the abovementioned limits.

In addition to this, the lid nanoparticles can also exhibit a pronouncedneedle-shaped morphology, with a breadth of from 3 nm to 50 nm,preferably of from 3 nm to less than 20 nm, and a length of from 20 nmto 5 μm, preferably of from 20 nm to 500 nm. In this case, breadth isunderstood as meaning the maximum separation of two points which lie onthe surface of a needle-shaped lid nanoparticle and, at the same time,in a plane which is perpendicular to the longitudinal axis of theneedle-shaped lid nanoparticle. The particle size can be determined bythe method of ultracentrifugation or of gel permeation chromatography orby means of electron microscopy.

Materials which are suitable, within the meaning of the invention, forthe lid nanoparticles are inorganic nanocrystals whose crystal lattices(host material) are doped with foreign ions. These materials include, inparticular, all the materials and material classes which are used aswhat are termed phosphors, e.g. in luminescent screens (e.g. forcathode-ray tubes) or as coating material in fluorescent lamps (forgas-discharge lamps), as are mentioned, for example, in Ullmann'sEncyclopedia of Industrial Chemistry, WILEY-VCH, 6^(th) edition, 2001Electronic Release, the “Luminescent Materials: 1. Inorganic Phosphors”chapter, and also the lid nanoparticles which are known from theabove-cited prior art. In these materials, the foreign ions serve asactivators for the emission of fluorescent light following excitation byUV light, visible light or IR light, X-rays or gamma rays, or electronbeams. In the case of some materials, several types of foreign ions arealso incorporated into the host lattice in order, on the one hand, toproduce activators for the emission and, on the other hand, to make theexcitation of the particle system more efficient or in order to adjustthe absorption wavelength by shifting it to the wavelength of a givenexcitation light source (what are termed sensitizers). The incorporationof several types of foreign ions can also be used to select a particularcombination of fluorescence bands which are to be emitted by a particle.

The host material of the lid nanoparticles preferably consists ofcompounds of the type XY. In this connection, X is a cation derived fromelements of the main groups 1a, 2a, 3a and 4a, of the subgroups 2b, 3b,4b, 5b, 6b or 7b, or of the lanthanides, of the periodic system. In somecases, X can also be a combination or mixture of said elements. Y can bea polyatomic anion which contains one or more element(s) of the maingroups 3a, 4a or 5a, or of the subgroups 3b, 4b, 5b, 6b, 7b and/or 8b,and also elements of the main groups 6a and/or 7a. However, Y can alsobe a monoatomic anion from the main group 5a, 6a or 7a of the periodicsystem. The host material of the lid nanoparticles can also consist ofan element from main group 4a of the periodic system. Elements of themain groups 1a and 2a, or from the group containing Al, Cr, Tl, Mn, Ag,Cu, As, Nb, Nd, Ni, Ti, In, Sb, Ga, Si, Pb, Bi, Zn and Co, and/orelements from the lanthanides, can be used as doping. Combinations oftwo or more of these elements can also be used, in different relativeconcentrations to each other, as doping material. The concentration ofthe doping material in the host lattice is between 10⁻⁵ mol % and 50 mol%, preferably between 0.01 mol % and 30 mol %, particularly preferablybetween 0.1 mol % and 20 mol %. The doping material is selected suchthat the decay time of the fluorescence which it induces is long (>100ns).

Sulfides, selenides, sulfoselenides, oxysulfides, borates, aluminates,gallates, silicates, germanates, phosphates, halophosphates, oxides,arsenates, vanadates, niobates, tantalates, sulfates, tungstates,molybdates, alkali metal halides and also other halides, or nitrides,are preferably used as host materials for the lid nanoparticles.Examples of these material classes are cited, together with thecorresponding dopings, in the following list (materials of the type B:A,with B=host material and A=doping material):

LiI:Eu; NaI:Tl; CsI:Tl; CsI:Na; Lif:Mg; LiF:Mg,Ti; LiF:Mg,Na; KMgF₃:Mn;Al₂O₃:Eu; BaFCl:Eu; BaFCl:Sm; BaFBr:Eu; BaFCl_(0.5)Br_(0.5):Sm; BaY₂F₈:A(A=Pr, Tm, Er, Ce); BaSi₂O₅:Pb; BaMg₂Al₁₆O₂₇:Eu; BaMgAl₁₄P₂₃:Eu;BaMgAl₁₀O₁₇:Eu; BaMgAl₂O₃:Eu; Ba₂P₂O₇:Ti; (Ba,Zn,Mg)₃Si₂O₇:Pb;Ce(Mg,Ba)Al₁₁O₁₉; Ce_(0.65)Tb_(0.35)MgAl₁₁O_(19:Ce,Tb); MgAl₁₁O₁₉Ce,Tb;MgF₂:Mn; ^(MgS:Eu; MgS:Ce; MgS:Sm; MgS:(Sm,Ce); (Mg,Ca)S:Eu;) MgSiO₃:Mn;3.5MgO.0.5MgF₂.GeO₂:Mn; MgWO₄:Sm; MgWO₄:Pb, 6MgO.As₂O₅:Mn; (Zn,Mg)F₂:Mn;(Zn₄Be)SO₄:Mn; Zn₂SiO₄:Mn; Zn₂SiO₄:Mn,As; ZnO:Zn; ZnO:Zn,Si,Ga;Zn₃(PO₄)₂:Mn; ZnS:A (A=Ag, Al, Cu); (Zn,Cd)S:Aa (A=Cu, Al, Ag, Ni);CdBO₄:Mn; CaF₂:Mn; CaF₂:Dy; CaS:A (A=lanthanides, Bi); (Ca,Sr)S:Bi;CaWO₄:Pb; CaWO₄:Sm; CaSO₄:A (A=Mn, lanthanides);3Ca₃(PO₄)₂.Ca(F,Cl)₂:Sb,M_(n); CaSiO₃:Mn,Pb; Ca₂Al₂Si₂O₇:Ce;(Ca,Mg)SiO₃:Ce; (Ca,Mg)SiO₃:Ti; 2SrO.6(B₂O₃).SrF₂:Eu;3Sr₃(PO₄)₂.CaCl₂:Eu; A₃(PO₄)₂.ACl₂:Eu (A=Sr, Ca, Ba); (Sr,Mg)₂P₂O₇:Eu;(Sr,Mg)₃(PO₄)₂:Sn; SrS:Ce; SrS:Sm,Ce; SrS:Sm; SrS:Eu; SrS:Eu,Sm;SrS:Cu,Ag; Sr₂P₂O₇:Sn; Sr₂P₂O₇:Eu; Sr4Al₁₄O₂₅:Eu; SrGa₂S₄:A(A=lanthanides, Pb); SrGa₂S₄:Pb; Sr₃Gd₂Si₆O₁₈:Pb,Mn; YF₃:Yb,Er; YF₃:Ln(Ln=lanthanides); YLiF₄:Ln (Ln=lanthanides; Y₃Al₅O₁₂:Ln(Ln=lanthanides); YAl₃(BO₄)₃:Nd,Yb; (Y,Ga)BO₃:Eu; (Y,Gd)BO₃:Eu;Y₂Al₃Ga₂O₁₂:Tb; Y₂SiO₅:Ln (Ln=lanthanides); Y₂O₃:Ln (Ln=lanthanides);Y₂O₂S:Ln (Ln=lanthanides); YVO₄:A (A=lanthanides, In); Y(P,V)O₄:Eu;YTaO₄:Nb; YAlO₃:A (A=Pr, Tm, Er, Ce); YoCl:Yb,Er; Ln1PO₄:Ln2 (Ln1,Ln2=lanthanides or mixtures of lanthanides); A_(x)(PO₄)_(y):Ln(A=alkaline earth metal, Ln=lanthanides) LuVO₄:Eu; GdVO₄:Eu; Gd₂O₂S:Tb;GdMgB₅O₁₀:Ce,Tb; LaOBr:Tb; La₂O₂S:Tb; LaF₃:Nd,Ce; BaYb₂F₈:Eu;NaYF₄:Yb,Er; NaGdF₄:Yb,Er; NaLaF₄:Yb,Er; LaF₃:Yb,Er,Tm; BaYF₅:Yb,Er;Ga₂O₃:Dy; GaN:A (A=Pr, Eu, Er, Tm); Bi₄Ge₃O₁₂; LiNbO₃:Nd,Yb; LiNbO₃:Er;LiCaAlF₆:Ce; LiSrAlF₆:Ce; LiLuF₄:A (A=Pr, Tm, Er, Ce); Li₂B₄O₇:Mn,SiO_(x):Er,Al (0≦x≦2).

The following materials are particularly preferably used as lidnanoparticles: YVO₄:Eu, YVO₄:Sm, YVO₄:Dy, LaPO₄:Eu, LaPO₄:Ce, LaPO₄:Tb,LaPO₄:Ce,Th, LaPO₄:Ce,Sm, LaPO₄:Ce,Dy, LaPO₄:Ce,Nd, ZnS:Tb, ZnS:TbF₃,ZnS:Eu, ZnS:EuF₃, Y₂O₃:Eu, Y₂O₂S:Eu, Y₂SiO₅:Eu, SiO₂:Dy, SiO₂:Al,Y₂O₃:Tb, CdS:Mn, ZnS:Tb, ZnS:Ag, ZnS:Cu. Those particularly preferredmaterials whose host lattice has a cubic structure are selected, inparticular, since, in the case of these materials, the number ofindividual fluorescence bands reaches a minimum. Examples of thesematerials are: MgF₂:Mn; ZnS:Mn, ZnS:Ag, ZnS:Cu, ^(CaSiO3:Ln,) CaS:Ln,CaO:Ln, ZnS:Ln, Y₂O₃:Ln, or MgF₂:Ln (Ln=lanthanides).

The surface of the lid nanoparticles which are used in accordance withthe invention is prepared in such a way that it is possible to coupleaffinity molecules to this prepared surface. In the assay according tothe invention, the affinity molecule enters into interaction with targetmolecules. Examples of affinity molecules are, e.g., proteins, peptidesor oligonucleotides or other nucleic acid molecules or nucleic acid-likemolecules, such as PNAs or morpholinos, or oligosaccharides orpolysaccharides or haptens, such as biotin or digoxin, or low molecularweight synthetic or natural antigens or epitopes.

The preparation of the surface of the lid nanoparticles can consist inthe surface of the lid nanoparticles being modified chemically and/orexhibiting reactive groups and/or connecting molecules which are boundcovalently or noncovalently. The connecting molecules which are bound tothe surface of the lid nanoparticles can also possess reactive groups.

These reactive groups, which can be charged, uncharged or provided withpartial charges, can be either on the surface of the lid nanoparticlesor be a part of the connecting molecules. Possible reactive functionalgroups can be amino groups, carboxylic acid groups, thiols, thioethers,disulfides, imidazoles, guanidines, hydroxyl groups, indoles, vicinaldiols, aldehydes, alpha-haloacetyl groups, N-maleimides, mercurides,aryl halides, acid anhydrides, isocyanates, isothiocyanates, sulfonylhalides, imidoesters, diazoacetates, diazonium salts, 1,2-diketones,alpha-beta-unsaturated carbonyl compounds, phosphonic acids, phosphoricesters, sulfonic acids or azolides, or derivatives of said groups.

Examples of connecting molecules which may be mentioned here arephosphonic acid derivatives, ethylene glycol, primary alcohols, aminederivatives, polymers or copolymers, polymerizable coupling agents,silica shells and catenate molecules having a polarity which is oppositeto that of the surface of the lid nanoparticles.

It is also possible to use nucleic acid molecules as connectingmolecules. They form the connection to an affinity molecule which, forits part, contains nucleic acid molecules possessing sequences which arecomplementary to the connecting molecules.

An affinity molecule can be bound to connecting molecules covalently ornoncovalently using standard methods of organic chemistry such asoxidation, halogenation, alkylation, acylation, addition, substitutionor amidation of the adsorbed or adsorbable material. These methods forbinding an affinity molecule to the covalently bound or noncovalentlybound connecting molecule can be employed before adsorbing theconnecting molecule to the lid nanoparticle or after the connectingmolecule has already been adsorbed to the lid nanoparticle. It is alsopossible to bind affinity molecules directly, by incubation, toappropriately treated (e.g. with trimethylsilyl bromide) lidnanoparticles, which, as a result of the treatment, possess a surfacewhich has been altered (e.g. more highly charged, polar).

The molecule groups A and B, which are labeled with donor or acceptor,can be constituents of one and the same molecule and can couple, forexample, to the same affinity molecule. A change in the spatialseparation of the two molecule groups can be brought about by a changein conformation or by the molecule being cleaved. This change inconformation or cleavage of the molecule can be the result of aninteraction of the shared affinity molecule with a target molecule.

The molecule groups A and B can also be located on different molecules,with the molecule groups A and B in each case being coupled to their ownaffinity molecules.

A spatial change in separation can be brought about by an interaction ofthe affinity molecules assigned to the molecule groups A and B with acommon target molecule or with each other. Such an interaction can, byway of example, comprise an interaction between proteins, e.g. an immunereaction of antigen and antibody, a hybridization of nucleic acids orthe interaction between nucleic acids and proteins.

The assay according to the invention can, for example, be a homogeneousimmunoassay for detecting an analyte (monoclonal or polyclonal antibody,protein, peptide, oligonucleotide, nucleic acid, oligosaccharide,polysaccharide, hapten or low molecular weight synthetic or naturalantigen) in a body sample (such as smears, sputum, organ punctates,biopsies, secretions, spinal fluid, bile, blood, lymphatic fluid, urineor feces). In homogeneous assays, no washing or separating steps arerequired.

The assay according to the invention can also be a heterogeneous assay.

The assay according to the invention can be employed in solution or insolid phase-supported or array-based systems in which oligonucleotide orpolynucleotide strands or antibodies or antigens are immobilized on asurface.

There are various groups of applications for the assay according to theinvention.

For one group of applications, the (F)RET partners are located on thesame molecule; this means that both (F)RET partners are bound to thesame affinity molecule by way of appropriate connecting molecules. Thebinding of a target molecule to the affinity molecule induces a changein the conformation of the affinity molecule, leading to a spatialchange in the labels and consequently to a measurable difference in the(F)RET between them.

For other applications, the (F)RET partners are located on separatemolecules and each is coupled to its own affinity molecule. Therespective affinity molecules can be selected such that the donor andacceptor interact, with this interaction being produced or terminated bythe reaction with the target molecule, thereby inducing a change in theenergy transfer.

As an example of a homogeneous kinase assay using (F)RET partners on thesame molecule, a lid nanoparticle and a chromophore are connected by apeptide sequence. The peptide sequence contains a kinase-specificrecognition sequence. If the peptide sequence is phosphorylated at thissite by the kinase, the presence of the phosphate alters theconformation of the peptide sequence and thereby alters, in a measurablemanner, the interaction between the (F)RET partners lid nanoparticle andthe chromophore.

As an example of a homogeneous immunoassay using (F)RET partners on onemolecule in which protein-protein interactions are detected, withantigen-antibody reactions only representing one example of this, a lidnanoparticle and a chromophore are linked by a peptide sequence. Thepeptide sequence contains an epitope. If an antibody, which specificallyrecognizes this epitope, binds to the epitope, this then alters theconformation of the peptide sequence and thereby alters, in a measurablemanner, the interaction between the (F)RET partners lid nanoparticle anda chromophore.

While the molecule to be detected can bind directly to the affinitymolecule, as described above, it can also be indirectly responsible fora molecule binding to the affinity molecule. An example of this would bethat of measuring Ca²⁺ concentrations in living cells. For this, use ismade of the calcium-dependent binding of calmodulin to the myosinlight-chain kinase (MLCK) in smooth muscles. The calmodulin-bindingdomain of MLCK acts as an affinity molecule and is linked to the (F)RETpartners. Depending on the Ca²⁺ concentration, calmodulin binds to thebinding domain and brings about a change in the conformation of thedetection probe and leads to a change in the measurable (F)RET.

As an example of a competitive immunoassay using (F)RET partners on onemolecule, which assay can be employed to detect the concentration of ananalyte in a body sample, a lid nanoparticle and a chromophore arelinked by a connecting molecule which contains the epitope. The epitopeis modelled on an epitope of the analyte to be detected. An affinitymolecule binds specifically to the epitope. Adding a sample (e.g. bodysample) which contains the analyte to be detected displaces the affinitymolecule, which is bound to the epitope, from the epitope, resulting ina change in the conformation of the molecule and, as a result, ameasurable change in the interaction between the (F)RET partners lidnanoparticle and the chromophore. This change in the (F)RET is used todetermine the concentration of the analyte.

As an example of a homogeneous saturation immunoassay using (F)RETpartners on separate molecules, the affinity molecules of the lidnanoparticle and the chromophore are able to recognize differentepitopes of the same target molecule, such that the presence of thetarget molecule results in a measurable energy transfer.

The detection of hCG (human chorionic gonadotrophin) in serum may bementioned here as an example of a homogeneous immunoassay in which donorand acceptor are located on separate molecules. In this assay, donor andacceptor are coupled to antibodies which recognize different hCGepitopes. If hCG is present in a body sample, both donor and acceptorprobes bind to the analyte. A calibration curve can be used to translatethe measurable FRET into a concentration of the analyte in the bodysample.

As an example of a homogeneous, competitive immunoassay using (F)RETpartners and on separate molecules, one or more chromophores are linkedto a molecule, which corresponds to parts of, or completely to, themolecule to be detected. A lid nanoparticle is coupled to an affinitymolecule which interacts specifically both with the molecule and withthe molecule to be detected. Binding occurs between the molecules,resulting in a (F)RET. If a sample (e.g. body sample) containing themolecule to be measured is now added, a displacement reaction takesplace depending on the concentration, in said sample, of the molecule tobe detected. This leads to a measurable change, in this case to adecrease, in the (F)RET, and a calibration curve can be used todetermine the concentration of the molecule to be detected.

As an example of a homogeneous assay using (F)RET partners on onemolecule, a lid nanoparticle and a chromophore are linked by a peptideas an affinity molecule. This peptide can be cleaved by an enzyme to bedetected. Following cleavage, no (F)RET any longer takes place.

An assay of this type can be used for detecting a particular enzymeactivity, for example a protease which is specific for the HI virus,within a sample or cell, with the two (F)RET partners being linked bythe short recognition sequence of this protease and being spatiallyseparated from each other by the activity of the protease, namelycleavage of the peptide. The enzyme activity to be detected can also bea restriction endonuclease. In this case, the two (F)RET partners arelinked by a nucleic acid.

As an example, the inventive assay can be conducted in accordance withthe molecular beacon method. Molecular beacons are DNA molecules whichare able to fold, by means of intramolecular complementary sequences,into what is termed a stem loop or hairpin structure. A lid nanoparticleis coupled to one end of the DNA sequence while a chromophore islocated, as fluorescence quencher, at the other end. In the hairpinstructure, the two (F)RET partners and are arranged closely adjacent toeach other and the fluorescence of the donor is therefore completelyquenched. The target molecule to be detected possesses sequences whichare complementary to the loop region of the DNA sequence. Since bindingto the target molecule is energetically more favorable, the hairpinconformation dissociates, chromophore and lid nanoparticle becomedetached from each other and fluorescence is measurably emitted since(F)RET no longer causes any fluorescence quenching. The hybridizationproperties can be adjusted such that just one single base mismatchbetween the molecular beacon and the target DNA results in the hairpinstructure not being open. As a result, it is even possible to detectsingle base differences (e.g. SNPs, single nucleotide polymorphisms).

EXAMPLES OF LINKING OF LID NANOPARTICLES TO ORGANIC MOLECULES Example 1Binding Phosphonic Acid Derivatives

The surface of the lid nanoparticle can be chemically modified, forexample, by binding on phosphonic acid derivatives which possessfunctional reactive groups. In this case, phosphonic acid derivatives orphosphonic ester derivatives, such asimino-bis(methylenephosphono)carboxylic acid (can be prepared, forexample, by means of the Mannich-Moedritzer reaction, Moedritzer andIrani, J. Org. Chem., 1966, 31, 1603) are bound stably to the surface ofthe lid nanoparticles. This binding can take place on untreated lidnanoparticles or on lid nanoparticles which have been previously treated(e.g. with trimethylsilyl bromide). Possible reactive functional groupswhich these phosphonic acid-containing or phosphonic ester-containingconnecting molecules carry can be amino groups, carboxylic acid groups,thiols, thioethers, disulfides, imidazoles, guanidines, hydroxyl groups,indoles, vicinal diols, anhydrides, isocyanates, isothiocyanates,sulfonyl halides, imidoesters, diazoacetates, diazonium salts,1,2-diketones, alpha-beta-unsaturated carbonyl compounds, phosphonicacids, phosphoric esters, sulfonic acids or azoles, or derivatives ofsaid groups.

Example 2 Binding of Ethylene Glycol, Primary Alcohols and Primary AmineDerivatives

Another example of treating the surface of the lid nanoparticles is thatof heating the particles in ethylene glycol, with the ethylene glycolbeing stably bound on the lid nanoparticles while the alkyl chains areeliminated. This treatment results in the particles becomingwater-soluble. It is possible to use primary alcohols possessingfunctionally reactive groups, as cited above, in an analogous manner. Ina similar way, primary amine derivatives can also be stably bound to thesurface of the lid nanoparticles. The amine derivatives can contain theabove-cited reactive functional groups in addition to the amine group.

Example 3 Coating with Silica, Polymers or Copolymers and PolymerizableCoupling Agents

The coating of a lid nanoparticle, or of a group of lid nanoparticles,with silica may be mentioned as another example of chemically modifyingthe surface of the lid nanoparticle: silica enables the nanoparticles tobe conjugated in a simple chemical manner to organic molecules sincesilica reacts very readily with organic linkers such as triethoxysilanesor chlorosilanes. Another example of a chemical modification is that ofcoating a lid nanoparticle, or a group of lid nanoparticles, withpolymers or copolymers. N-(3-Aminopropyl)-3-mercaptobenzamidines,3-(trimethoxysilyl)propyl hydrazide and3-(trimethoxysilyl)propylmaleimide may be mentioned as examples ofpolymerizable coupling agents. It is also possible to use a multiplicityof different polymerizable coupling agents together for the purpose ofproviding one or more lid nanoparticles with a coating layer. This canbe of particular advantage in the case of coupling agents which onlybind weakly to the lid nanoparticle. Examples of coupling agents whichare able to form such networks, as the coating for one or more lidnanoparticles, are diacetylenes, styrenebutadienes, vinyl acetate,acrylates, acrylamides, vinyls, styrenes, silicone oxides, boron oxides,phosphorus oxides, borates, pyrroles, polypyrroles and phosphates, andalso polymers of at least some of said agents.

Example 4 Surface Modification Using Oxychlorides, and also NoncovalentLinkages Using Catenate Molecules and Amphiphilic Reagents

Another option for preparing the surface of the lid nanoparticles isthat of using chlorine gas, or organic chlorinating agents, to convertthe oxidic transition metal compounds, of which the lid nanoparticlesconsist, into the corresponding oxychlorides. These oxychloridesthemselves react with nucleophiles, such as amino groups, with theformation of transition metal nitrogen compounds. In this way, it ispossible, for example, to achieve direct conjugation of proteins via theamino groups of lysine side chains. Conjugating to proteins, after thesurface modification with oxychlorides, can also be effected by using abifunctional linker such as maleimidopropionic acid hydrazide.

In this connection, catenate molecules having a polarity or charge whichis opposite to that of the surface of the lid nanoparticle areparticularly suitable for noncovalent linkages. Examples of connectingmolecules which are linked noncovalently to the lid nanoparticles, andwhich may be mentioned, are anionic, cationic or zwitterionicdetergents, acid or basic proteins, polyamines, polyamides, polysulfonicacids and polycarboxylic acids. It is possible to create a linkage bymeans of hydrophobic interaction between the lid nanoparticles andamphiphilic reagents which carry a functional reactive group. Chainmolecules which have an amphiphilic character, such as phospholipids orderivatized polysaccharides, which can be crosslinked to each other areparticularly suitable for this purpose. These molecules can be adsorbedon the surface of the lid nanoparticle by means of coincubation.

Example 5 Using a Connecting Molecule to Bind Affinity Molecules to LidNanoparticles

In general, it is possible to use the same affinity molecules as thosewhich are also employed in the case of the fluorescent organic dyemolecules which are described in the prior art for specifically bindingthese latter molecules to the biological, or other organic, substancewhich is to be detected. An affinity molecule can be a monoclonal orpolyclonal antibody, a different protein, a peptide, an oligonucleotide,a plasmid or another nucleic acid molecule, a peptide nucleic acid (PNA)or a morpholino, an oligosaccharide, a polysaccharide, or a hapten, suchas biotin or digoxin, or a low molecular weight synthetic or naturalantigen. A list of these molecules are published in the generallyavailable literature, e.g. in the “Handbook of Fluorescent Probes andResearch Chemicals” (8^(th) edition, 2001, CD-ROM) by R. P. Hauglund,Molecular Probes, Inc.

Reactive groups on the surface of the affinity molecule are used forbinding affinity molecules covalently or noncovalently to the lidnanoparticles using a connecting molecule. In this connection, reactivegroups on the surface of the affinity molecule are amino groups,carboxylic acid groups, thiols, thioethers, disulfides, imidazoles,guanidines, hydroxyl groups, indoles, vicinal diols, aldehydes,alpha-haloacetyl groups, N-maleimides, mercurides, aryl halides, acidanhydrides, isocyanates, isothiocyanates, sulfonyl halides, imidoesters,diazoacetates, diazonium salts, 1,2-diketones, alpha-beta-unsaturatedcarbonyl compounds, phosphonic acids, phosphoric esters, sulfonic acidsor azolides, or derivatives of said groups. On the surface of the lidnanoparticles, it is possible to use the functional reactive groups,which were described earlier on, of the connecting molecules forconjugating the affinity molecule. Protocols for effecting couplings toreactive groups are described in the generally available literature,e.g. in “Bioconjugate Techniques” (by Greg T. Hermanson, Academic Press1996).

If donor and acceptor are present on the same molecule, the affinitymolecule, which is coupled to the donor by way of one or more connectingmolecules, can be bound covalently or noncovalently to the acceptor.Chromophores whose excitation wavelength overlap with the emissionwavelength of the respective donor can be used as acceptor. Acceptorscan, for example, be lid nanoparticles, organic dyes (e.g. fluorescein,rhodamine, cyanines), organic pigments (e.g. perylenes) or conductingmaterials (e.g. metals, doped oxides, conducting polymers). Thesematerials can either be present as particular systems or as a planar orstructured surface or surface coating. The coupling can take place byway of reactive groups on the affinity molecule and/or the acceptormolecule.

In addition to affinity molecules being linked covalently, it is alsopossible to produce noncovalent, self-organized compounds. Onepossibility which may be mentioned in this context is the linking ofsimple detection probes to avidine-coupled or streptavidine-coupledaffinity molecules using biotin as the connecting molecule.

Example 6 Preparing Lid Nanoparticles Composed of YVO₄:Eu

The first step consists in preparing YVO₄:Eu. YVO₄:Eu can be preparedusing the method given in K. Riwotzki, M. Haase; Journal of PhysicalChemistry B; Vol. 102, 1998, page 10130, left-hand column: 3.413 g ofY(NO₃)₃.6H₂O (8.9 mmol) and 0.209 g of Eu(NO₃)₃.6H₂O (0.47 mmol) aredissolved in 30 ml of distilled water in a Teflon receptacle. 2.73 g ofNa₃(VO₄).10H₂O, dissolved in 30 ml of distilled water, are added to thissolution, while stirring. After a further 20 min of stirring, the Teflonreceptacle is placed in an autoclave and heated at 200° C. whilecontinuing to be stirred. After 1 h, the dispersion is removed from theautoclave and centrifuged at 3000 g for 10 min, and the supernatant isdiscarded. The precipitate is taken up in 40 ml of distilled water.3.220 g of an aqueous solution of 1-hydroxyethane-1,1-diphosphonic acid(60% by weight) (9.38 mmol) are added to the dispersion. In order toremove Y(OH)₃, which has formed from excess yttrium ions, the pH isadjusted to 0.3 with HNO₃ and the mixture is stirred for 1 h. ColloidalV₂O₅, which becomes apparent as a result of the solution assuming areddish color, is formed in this connection. After that, the pH isadjusted to 12.5 with NaOH and the mixture is stirred overnight in aclosed receptacle. The resulting white dispersion is then centrifuged at3000 g for 10 min and the supernatant, together with its by-products, isremoved. The precipitate consists of YVO₄:Eu and can be taken up in 40ml of distilled water.

In order to isolate the nanoparticles, which are smaller than approx. 30nm, the dispersion is centrifuged at 3000 g for 10 min, after which thesupernatant is decanted and kept on one side. The precipitate is thentaken up once again in 40 ml of distilled water and this mixture iscentrifuged at 3000 g for 10 min, after which the supernatant isdecanted. This supernatant, and the supernatant which is kept on oneside, are combined and centrifuged at 60 000 g for 10 min. Thesupernatant resulting from this centrifugation contains the desiredparticles. A colloidal solution, from which a redispersible powder canbe isolated by drying with a rotary evaporator (50° C.), is obtainedafter a further step of dialysis against the distilled water (dialysistubing from Serva, Heidelberg, MWCO 12-14 kD).

Example 7 Preparing Lid Nanoparticles Composed of LaPO₄:Eu³⁺

The first step consists in preparing LaPO₄:Eu. LaPO₄:Eu can be preparedusing the method given in H. Meyssamy, K. Riwotzki, A. Kornowski, S.Naused, M. Haase; Advanced Materials, Vol. 11, Issue 10, 1999, bottom ofright-hand column on page 843 to top of left-hand column on page 844:12.34 g of La(NO₃)₃.6H₂O (28.5 mmol) and 0.642 g of Eu(NO₃)₃.5H₂O (1.5mmol) are dissolved in 50 ml of distilled water and added to 100 ml ofNaOH (1 M) in a Teflon receptacle. A solution of 3.56 g (NH₄)₂HPO₄ (27mmol) in 100 ml of distilled water is added to this mixture whilestirring. The solution is adjusted to pH 12.5 with NaOH (4 M) and thenheated at 200° C. for 2 h in an autoclave while being stirredvigorously. The dispersion is then centrifuged at 3150 g for 10 min andthe supernatant is removed. In order to remove unwanted La(OH)₃, theprecipitate is dispersed in HNO₃ (1M) and stirred for 3 days (pH 1).After that, the dispersion is centrifuged (3150 g, 5 min) and thesupernatant is removed. 40 ml of distilled water are added to thecentrifugate while stirring.

The milky dispersion still contains a broad size distribution. In orderto isolate the nanoparticles which are smaller than approx. 30 nm,appropriate centrifugation and decantation steps are subsequentlyperformed in complete analogy with Example 6.

Example 8 Preparing Lid Nanoparticles Composed of LaPO₄:Ce, Tb

The first step consists in preparing LaPO₄:Ce,Tb. 300 ml oftrisethylhexyl phosphate are degassed with a dry stream of nitrogen gas.7.43 g of LaCl₃.7H₂O (20 mmol), 8.38 g of CeCl₃.7H₂O (22.5 mmol) and 2.8g of TbCl₃.6H₂O (7.5 mmol) are then dissolved in 100 ml of methanol andadded. After that, water and methanol are distilled off in vacuo byheating the solution to from 30° C. to 40° C. A freshly preparedsolution consisting of 4.9 g of dry orthophosphoric acid (50 mmol),which have been dissolved in a mixture of 65.5 ml of trioctylamine (150mmol) and 150 ml of trisethylhexyl phosphate, are then added. The clearsolution has to be rapidly placed in a vessel which can be evacuated,and flushed with a stream of nitrogen gas, in order to minimize theoxidation of Ce³⁺ when the temperature is raised. The solution issubsequently heated at 200° C. During the heating phase, some of thephosphoric acid ester groups are cleaved, leading to a gradual loweringof the boiling point. The heating phase is terminated when thetemperature falls to 175° C. (from approx. 30 to 40 h). After thesolution has cooled down to room temperature, a 4-fold excess ofmethanol is added, leading to the nanoparticles precipitating out. Theprecipitate is separated off, washed with methanol and dried.

Example 9 Preparing Lid Nanoparticles Composed of LaPO₄:Eu³⁺

490 mg (5.0 mmol) of dry orthophosphoric acid and 6.5 ml (15 mmol) oftrioctylamine are dissolved in 30 ml of trisethylhexyl phosphate. 1.76 gof La(NO₃)₃.7H₂O (4.75 mmol) and 92 mg of EuCl₃.6H₂O (0.25 mmol) arethen dissolved in 50 ml of trisethylhexyl phosphate and this solution iscombined with the first solution. The resulting solution is degassed invacuo and then heated at 200° C. for 16 h under nitrogen. During theheating phase, some of the phosphoric acid ester groups are cleaved,leading to a gradual lowering of the boiling point. The heating phase isterminated when the temperature falls to 180° C. After the solution hascooled down to room temperature, methanol is added, leading to thenanoparticles precipitating out. The precipitate is separated off usinga centrifuge, washed twice with methanol and dried.

Example 10 Dissolving the Nanoparticles Prepared in Example 8 in Waterby the Reaction of Ethylene Glycol or Polyethylene Glycol

50 mg of the LaPO₄:Ce,Tb nanoparticles (˜140 nmol) prepared in Example 8are heated, at 210° C. for 3 hrs and while stirring and under an inertgas, with 5 ml of ethylene glycol (˜180 mmol) (as an alternative, it isalso possible to use other alcohols, in particular diols, preferablypolyethylene glycols of differing chain length, HO—(CH₂—CH₂—O)_(n)—OH,where n=2-9) and 5 μl of sulfuric acid (96-98%). The particles go intosolution at approx. 135° C. A water jet vacuum of approx. 1.5 mbar isthen applied and about half the ethylene glycol is distilled off; theresidue remains clear. The residue is then dialyzed against waterovernight (Spectra/Por dialysis tubing, 5-6,000 MWCO, Spektrum,Netherlands).

Example 11 Using Oxidation to Carboxyl-functionalize NanoparticlesPrepared in Example 10

0.5 ml of 96-98% strength sulfuric acid is first of all added, whilestirring, to 100 mg (˜300 nmol) of the nanoparticles prepared in Example10 in 20 ml of water. 1 mM KMnO₄ solution is added dropwise until thereis no further decoloration of the violet color. The same quantity ofKMnO₄ solution is then added once again and the mixture is left to stirovernight (>12 h) at room temperature. Excess permanganate is reduced byadding freshly prepared 1 mM sodium sulfite solution dropwise. Themixture is dialyzed overnight against 0.1M MES, 0.5M NaCl, pH 6.0(Spectra/Por dialysis tubing, 5-6,000 MWCO, Spektrum, Netherlands).

Example 12 Removing the Alkyl Chains of the Trisethylhexyl Phosphatefrom the Surface of the Nanoparticles Described in Example 8 by Means ofReaction with Bromotrimethylsilane

300 mg of the LaPO₄:Ce,Tb nanoparticles (˜850 nmol) prepared in Example8 are boiled, under reflux for 4 hours, with 2.3 g (15 mmol) ofbromotrimethylsilane in 100 ml of chloroform; most of thebromotrimethylsilane excess, and the intermediate which is formed, aredistilled off and hydrolysis with a low concentration of ammoniasubsequently takes place. For this, 100 μl of 25% strength ammonia areadded to 6 ml of water and stirring takes place at RT overnight. Theparticles are present in a milky emulsion, and a portion of themsedimented out after several hours.

Example 13 Coupling the Nanoparticles Prepared in Example 12 to11-bis-(phosphorylmethyl)aminoundecanoic acid and1,4-bis(3-aminopropoxy)butane

In order to prepare 11-bis(phosphorylmethyl)aminoundecanoic acid, 201 gof 11-aminoundecanoic acid, 170 g of phosphorous acid, 200 ml ofconcentrated hydrochloric acid and 200 ml of water are initiallyintroduced and heated to 100° C. 324 g of formalin (37%) are then addeddropwise within the space of 1 h and the mixture is subsequently stirredat 100° C. for a further 1 h. After the mixture has cooled down to roomtemperature, the product is filtered off with suction and dried invacuo. This results in 334 g of 11-bis(phosphorylmethyl)aminoundecanoicacid, which was characterized by means of elemental analysis (C,H,N,P).

As an alternative, it is possible to use molecules of the followingformula, where the radical (R) is an alkylene having from 1 to 17 carbonatoms, preferably 3-12 carbon atoms, or an alkylenearylene radicalhaving 7-12 carbon atoms:

0.894 g (4.375 mmol) of 1,4-bis(3-aminopropoxy)butane is added to 0.5 g(1.85 mmol) of 11-bis(phosphorylmethyl)aminoundecanoic acid in 2 ml ofethylene glycol. After a clear solution has formed (exothermicreaction), 35 mg (=100 nmol) of the nanoparticles prepared in Example 12are added at approx. 50° C. and the mixture is heated to 125° C. Theparticles go into solution completely at 120° C. After 4 hrs, a clearlight brown solution is present, with this solution also remaining clearafter it has cooled down to RT. It is dialyzed overnight against 2×2 lof 10 mM Na carbonate buffer, pH 8.5 (Spectra/Por dialysis tubing,5-6,000 MWCO, Spektrum, Netherlands). The dialysate is clear.

Example 14 Biotinylating Nanoparticles Prepared in Example 13

6.2 ml (=5 mg or ˜15 nmol) of the particles prepared in Example 13 areinspissated on a rotary evaporator and concentrated down to 4.81 mg/ml.The particles are incubated, at RT for 4 hrs and while being rotated,with a 20-fold molar excess of biotin-X-NHS (sulfobiotin-aminocaproicacid-N-hydroxysuccinimide ester, Calbiochem, Schwalbach), which isdissolved in water, and then dialyzed against PBS buffer (8 mM K₂HPO₄;150 mM NaCl; 2 mM Na₂HPO₄; pH 7.4) (Spectra/Por dialysis tubing, 5-6,000MWCO, Spektrum, Netherlands). The dialysate is slightly turbid.

Example 15 Coupling a DNA Oligonucleotide to the Nanoparticles Preparedin Example 13

The nanoparticles prepared in Example 13 are activated with a 40-foldexcess of sulfo-SIAB (sulfosuccinimidyl (4-iodoacetyl)aminobenzoate,Perbio Science Deutschland GmbH, Bonn); 7.5 mg of aminofunctionalizednanoparticles (˜25 nmol) are rebuffered in TSMZ buffer, pH 7.3 (0.1 MNaCl; 0.1 M triethanolamine-HCl; 0.02 M NaOH; 0.27 mM ZnCl₂; 0.1% Tween20; 1 mM MgCl₂) using a Centricon filter unit (MW exclusion limit at 50000, Millipore, Eschborn) and adjusted to a concentration of about 7mg/ml. 50 μl of a 20 mM solution of sulfo-SIAB in water are added to theparticle solution and the whole is incubated at 25° C. for 15 mm. Thereaction is stopped by adding 12 μl of 1 M glycine (12-fold excess) andthe free sulfo-SIAB is separated off through a ready-to-use Sephadex G25PD 10 column (Amersham Pharmacia Biotech, Freiburg). A DNAoligonucleotide having the sequence 5′-CCACGCTTGTGGGTCAACCCCCGTGG-3′(SEQ ID NO: 1), and with a thiol modification at the 5′ end and a dabycl(4-(4-dimethylaminophenylazo)benzoyl) modification at the 3′ end, andalso a control DNA oligonucleotide, which only differs in lacking thedabcyl molecule at the 3′ end of the probe, were obtained fromInteractiva (Ulm). Equimolar quantities of the DNA oligonucleotide andthe SIAB-activated nanoparticles are mixed and incubated at 25° C. for 3hrs and then incubated at 4° C. overnight. The DNAoligonucleotide-coupled particles are separated off from uncoupledparticles and free DNA oligonucleotide by means of an FPLC (fastperformance liquid chromatography). The coupled particles are taken upin 50 mM tris-HCl, pH 7.4; 0.1% BSA at 4° C. If no target DNA isavailable, this molecule is present in a hairpin structure, with theends of the molecule being directly adjacent, and a FRET takes place.The fluorescence of the nanoparticle in this connection is quenched bydabcyl.

Example 16 Coupling Anti-β-hCG Monoclonal Antibody to the NanoparticlesPrepared in Example 13

The particles prepared in Example 13 are first of all activated with a30-fold molar excess of 2-iminothiolane (2-IT, Traut's reagent, PerbioScience Deutschland GmbH, Bonn): 2 ml (˜25 nmol) of the particlesprepared in Example 13 (4 mg/ml) are transferred into TSE buffer, pH 8.5(0.04 M NaCl; 0.05 M triethanolamine-HCl; 0.04 M NaOH; 0.5 mM EDTA; 0.1%Tween 20; pH 8.5). For this, the particles are centrifuged at 3000 g for3×15 mm and, after the excess has been decanted off, the sediment is ineach case taken up in 700 μl of TSE buffer, pH 8.5. These particles areincubated, at 25° C. for 1 h, with 75 μl of 10 mM 2-IT (in TSE buffer,pH 8.5) and the reaction is then stopped with 9 μl (12-fold excess) of 1M glycine. In order to separate off the excess of 2-IT, the mixture isonce again centrifuged at 3000 g for 3×15 mm and, after decanting off,the sediment is resuspended twice in 1 ml of TSE buffer, pH 7.3 (0.1 MNaCl; 0.1 M triethanolamine-HCl; 0.02 M NaOH; 1 mM EDTA; 0.1% Tween 20;pH 7.3) and, after the third centrifugation, in 250 μl of TSE buffer, pH7.3. At the same time, an equimolar quantity of the β-hCG-specific mousemonoclonal antibody (clone F199C1, Perkin-Elmer Life Sciences—Wallac Oy,Finland) is activated with a 40-fold excess ofSMCC(N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,Perbio Science Deutschland GmbH, Bonn); 750 μl of anti-β-hCG antibody(=25 nmol at a concentration of 5 mg/ml) are rebuffered in TSMZ buffer,pH 7.3 (0.1 M NaCl; 0.1 M triethanolamine-HCl; 0.02 M NaOH; 0.27 mMZnCl₂; 0.1% Tween 20; 1 mM MgCl₂) using a Centricon filter unit(molecular weight exclusion limit at 50 000) and the solution isadjusted to a concentration of 7 mg/ml. 50 μl of a 20 mM solution ofSMCC in DMF (=11 mmol) are added to the antibody solution and themixture is incubated at 25° C. for 30 min. The reaction is stopped byadding 12 μl of 1 M glycine (12-fold excess) and the free SMCC isseparated off through a ready-to-use Sephadex G25 PD 10 column (AmershamPharmacia Biotech, Freiburg). Finally, equimolar quantities of the2-IT-activated particle solution and the SMCC-activated antibodysolution are mixed and this mixture is incubated at 25° C. for 3 hrs andthen at 4° C. overnight. The antibody-coupled particles are purifiedfrom noncoupled particles and free antibody by means of gel permeationchromatography on Superdex 200 (Amersham Pharmacia Biotech, Freiburg).0.1M MES, 0.5M NaCl, pH 6.0, is used as the running buffer. Theretention time for the enlarged lid nanoparticles is about 2 hours.

Example 17 Coupling the LaPO4: Eu³⁺ Nanoparticles Prepared in Example 9to hIL-2

300 mg of the LaPO4:Eu³⁺ nanoparticles (˜1 μmol) prepared in Example 9are boiled, under reflux and for 4 hours, with 2.23 g (15 mmol) ofbromotrimethylsilane in 125 ml of chloroform; most of thebromotrimethylsilane excess and the intermediate which is formed aredistilled off and hydrolysis subsequently takes place with a lowconcentration of ammonia. For this, 100 μl of 25% strength ammonia areadded to 6 ml of water, with stirring then taking place at RT overnight.The particles are present in a milky emulsion and a portion of themsettled out after several hours. 5 mg (=25 mmol, 106 μl) of thesebromotrimethylsilane-treated nanoparticles are incubated, at 37° C. for1 h and while shaking, with recombinant human IL-2 protein (R&D Systems,Minneapolis, Minn., USA) in 10 mM Na carbonate buffer, pH 8.5, in amolar ratio of 2:1. The excess protein is subsequently separated off bycentrifuging 6 times at 3000 g for 10 min and in each case resuspendingin 1 ml of 10 mM Na carbonate buffer, pH 8.5. The LaPO₄:Eu³⁺/IL-2conjugate is stored at 4° C.

Example 18 Homogeneous Energy Transfer Assay for Detecting β-hCG UsingAntibody-coupled Nanoarticles Prepared in Example 16 andFluorescein-coupled Antibodies as Acceptor

Coupling Anti-β-hCG Antibody to Fluorescein:

The Molecular Probes Fluoreporter® FITC protein labeling kit is used, inaccordance with the manufacturer's instructions, to couple theanti-β-hCG antibody (M15294, Perkin-Elmer Life Sciences, Wallac Oy,Finland) to fluorescein. 0.5 mg of the antibody is rebuffered in 0.2 Mhydrogen carbonate buffer, pH 9.0, using a Centricon filter unit(molecular weight exclusion limit 50 000). A 25-fold excess of a 5 mMsolution of fluorescein isothiocyanate (FITC) (dissolved in a mixture ofequal volume units of DMF and 0.2 M hydrogen carbonate buffer, pH 9.0)is then added to the antibody solution and the whole is incubated at RTfor 3 hrs. The excess of FITC is separated off through a ready-to-useSephadex G25 PD 10 column (Amersham Pharmacia Biotech, Freiburg) and theantibody concentration and the fluorescein/antibody ratio are determinedspectroscopically. 0.01% sodium azide and 0.1% BSA are added to theconjugate and the mixture is stored at 4° C.

Implementing the Assay:

50 μl of a β-hCG standard from a commercially available kit formeasuring free β-hCG in serum (A007-101, Perkin-Elmer Life Sciences,Wallac Oy, Finland), together with 100 nmol of the nanoparticle-antibodyconjugate prepared in Example 16 and 100 mmol of the fluorescein-coupledanti-β-hCG antibody, are incubated, at 25° C. for 60 min, in 200 μl oftris-HCl buffer, pH 7.4, in a UV-permeable 96-well microtiter plate(UVStar, Greiner). The two anti-β-hCG antibodies are directed againstdifferent epitopes in the β-hCG subunit. The samples are subsequentlymeasured in a fluorescence spectrometer (from Jobin Yvon, Fluorolog 3)using the following settings: pulsed excitation with excitationwavelength: 280 nm, emission: 542 nm, slit width: 4 nm, time delay: 50μs, repetition rate, approx. 25 Hz. The half-life of the terbiumemission line can also be determined. The following settings are usedfor this purpose: excitation: 280 nm, emission 542 nm, slit width 5 nm;integration time: 0.1 ms. These measurements which are obtained areplotted against the β-hCG concentrations employed in order to constructa calibration curve. The body sample content of β-hCG can be measured inserum samples in an analogous manner and the concentration can bedetermined using the calibration curve.

Example 19 Homogeneous Competitive Energy Transfer Assay for DetermininghIL-2 Using hIL-2-Coupled Nanoparticles Prepared in Example 17 and AlexaFluor 680-Coupled anti-hIL-2Rα Chain Antibodies

Coupling the anti-hIL-2Rα Chain Monoclonal Antibody to Alexa Fluor 680:

1 mg of the monoclonal antibody 7G7B6, which specifically recognizes theα chain of the human interleukin-2 receptor (hIL-2Rα chain) (ATCC,Rockville, USA), is dialyzed against PBS, adjusted to a concentration of2 mg/ml and labeled using the Alexa Fluor 680 protein labeling kit(Molecular Probes Europe BV, Netherlands) in accordance with themanufacturer's instructions. 0.1 M Na bicarbonate buffer, pH 8.3, isused as the reaction buffer and the mixture is incubated at RT for 1 h.The coupled antibody is purified using a column contained in the kit,with PBS buffer containing 0.2 mM Na azide being employed as the elutionbuffer. In order to determine the protein concentration of the coupledantibody, the absorption (A) is measured at 280 and 679 nm in a 1 cmcuvette and the calculation uses the following formula:

$M = \frac{\left( {A_{280} - \left( {A_{679} \times 0\text{.}05} \right)} \right) \times \text{dilution}\mspace{14mu}{factor}}{203000}$where 203 000 cm⁻¹M⁻¹ is the molar extinction coefficient of an IgG and0.05 is the factor for correcting the absorption of the dye at 280 nm.The concentration of the coupled antibody is 1.27 and is adjusted withPBS; 0.2 mM Na azide to 1 mg/ml (˜6.5 μM); the antibody solution is thenstored at 4° C. The efficiency of the labeling is calculated as follows:

${{mol}\mspace{14mu}{of}\mspace{14mu}{dye}\mspace{14mu}{per}\mspace{14mu}{mol}\mspace{14mu}{of}\mspace{14mu}{antibody}} = \frac{A_{679} \times {dilution}\mspace{14mu}{factor}}{18000 \times {protein}\mspace{14mu}{concentration}\mspace{14mu} M}$where 184 000 cm⁻¹M⁻¹ is the molar extinction coefficient of the AlexaFluor 680 dye at 670 nm. The ratio of the antibody-dye conjugate is 3.2.Implementing the Assay:

The necessary dilutions of the different components are performed in 50mM TSA buffer (50 mM tris-HCl, pH 7.75; 0.9% NaCl; 0.05% NaN₃). 40 wellsof a UV-permeable microtiter plate (UVStar, Greiner) are first of allincubated, at RT for 1 h, with a 0.5% strength solution of BSA in orderto saturate nonspecific binding sites and then loaded with a mixtureconsisting of nanoparticles prepared in Example 17 (LaPO₄: Eu³⁺/IL-2conjugate), Alexa Fluor 680-labeled anti-hIL-2Rα chain antibody andrecombinant hIL-2sRα protein (human IL-2 soluble alpha receptor, R&DSystems, Minneapolis, Minn.) at a final concentration of 40 nM in eachcase. Unlabeled hIL-2 protein is added to 20 of the wells at differentconcentrations, while a protein which is irrelevant to this assay isadded to the other 20 wells. The concentration is in each case increasedby 50 nM such that a concentration series of 0-950 nM is tested. Thefinal volume of the reaction is in each case 200 μl. The plate isincubated at RT for 45 mm in the dark on a shaker. The signals are readusing a Wallac 1420 Victor™ multilabel counter (Perkin-Elmer LifeSciences Wallac Oy, Finland) and employing the following settings:excitation: 340 nm, emission: 665 nm, time delay: 50 μs, time window:200 μs and cycling time: 1000 μs. A duplicate determination is carriedout for each value and a correction is made for nonspecific bindingsites using the results obtained with the irrelevant protein. Themeasured values are plotted in a graph against the protein concentrationemployed and result in a calibration curve which can be used todetermine the concentrations of human interleukin-2 which are measured,in an analogous manner, in human body samples.

Example 20 Quantitative PCR Determination of Bacterial DNA by Means ofan Intramolecular Energy Transfer Using the Nanoparticles Prepared inExample 15

The primers and the probe for the quantitative DNA determination werechosen specifically for the Mycobacterium tuberculosis RNA polymerasegene and were prepared at Interactiva (Ulm). The primers have thefollowing sequences:

forward: 5′-GGCCGGTGGTCGCCGCG-3′, (SEQ ID NO: 2) backward:5′-ACGTGACAGACCGCCGGGC-3′. (SEQ ID NO: 3)Assay for Quantitatively Determining Bacterial DNA

50 nM of the nanoparticles prepared in Example 15(Dabcyl-oligonucleotide-coupled), as probe, in each case 500 nM of thetwo primers, 2 U of Amplitaq Gold DNA polymerase (Perkin-Elmer), 250 μMof dATP, 250 μM of dCTP, 250 μM of dGTP, 500 μM of dUTP, 4 mM of MgCl2,50 mM of KCl and 10 mM of tris-HCl, pH 8.0, are mixed for the 50 μL PCRreactions. As the DNA template, genomic M. tuberculosis DNA is amplifiedwith the same primers and cloned into a plasmid using the InvitrogenZero Blunt TOPO PCR Cloning Kit (Invitrogen BV/NOVEX, Netherlands). Inorder to construct a standard curve, 5 different concentrations, of from1 μg to 100 ng, of the DNA plasmid are used, as is a reaction withoutDNA template. 30 reactions are prepared for each concentration, suchthat, starting from the 15^(th) cycle, a sample can be withdrawn, formeasuring in a spectrometer, after each further cycle. The reactionvolume is 50 μL and the amplification is carried out on a Thermocycler(PCR system 2400, Perkin-Elmer) under the following reaction conditions:10 min, 95° C.; 15-45 cycles of 30 s at 95° C., 45 s at 56° C. and 30 sat 72° C. The samples are measured in a fluorescence spectrometer (fromJobin Yvon, Fluorolog 3) using the following settings: pulsed excitationwith excitation wavelength: 280 μm, emission: 542 nm, slit width: 4 nm,time delay: 50 μs, repetition rate approx. 25 Hz. It is also possible todetermine the half-life of the terbium emission line. The followingsettings are used for this purpose: excitation: 280 nm, emission 542 nm,slit width 5 nm; integration time: 0.1 ms. No intramolecular FRET takesplace between the nanoparticle and the dabcyl during hybridization ofthe probe to the target DNA. The Th fluorescence of the nanoparticletherefore becomes stronger, as compared with the control withouttemplate, as the concentration of the target DNA increases; at the sametime, the half-life of the fluorescence lifetime of the nanoparticlebecomes longer as compared with the control without template DNA. Thesedifferences in the two parameters can be plotted against the number ofcycles in order to construct a calibration curve for each DNA templateconcentration.

1. An assay kit for carrying out an assay method based on a resonanceenergy transfer (RET), said assay kit comprising: a) a first moleculegroup A which is labeled with at least one energy donor (donor), and b)at least a second molecule group B which is labeled with at least oneenergy acceptor (acceptor), wherein the donor comprises a molecule orparticle which can be energetically excited by an external radiationsource and is capable of fluorescing, the acceptor comprises a moleculeor particle which can be excited by energy transfer by way of the donor,with partial or complete quenching of the donor fluorescence, and donorand/or acceptor comprise luminescent inorganic doped nanoparticles (lidnanoparticles) which have a breadth of >50 nanometers and which, afteran energetic excitation, emit electromagnetic radiation with a Stokesshift or an anti-Stokes shift.
 2. The assay kit as claimed in claim 1,wherein the acceptor is also capable of fluorescing.
 3. The assay kit asclaimed in claim 1, wherein the acceptor relaxes in a radiationlessmanner.
 4. The assay kit as claimed in claim 1, wherein the RET is afluorescence resonance energy transfer (FRET).
 5. The assay kit asclaimed in claim 1, wherein the RET is a Förster transfer or a transferinvolving higher multiple orders.
 6. The assay kit as claimed in claim1, wherein the RET is a migration of charges or excitons.
 7. The assaykit as claimed in claim 1, wherein the RET can be qualitatively and/orquantitatively recorded by a time-resolved or continuous measurement ofa change in the luminescence properties.
 8. The assay kit as claimed inclaim 7, wherein the RET is qualitatively and/or quantitatively recordedby a time-resolved or continuous measurement of a change in an intensityof the fluorescent light, a change in the spectrum of the fluorescentlight or a change in the decay time of the lid nanoparticles and/or ofother donors and/or acceptors.
 9. The assay kit as claimed in claim 7,wherein the RET is qualitatively and/or quantitatively recorded by atime-resolved or continuous measurement of a change in an intensity ofthe fluorescent light, a change in the spectrum of the fluorescent lightor a change in the decay time of a chromophoric donor/acceptor.
 10. Theassay kit as claimed in claim 1, wherein either the donor or acceptorcomprises lid nanoparticles which have a long fluorescence decay timeand the other one of the donor or acceptor either also comprises lidnanoparticles whose fluorescence decay time is shorter than that of theother lid nanoparticles or comprises a molecular organic chromophore.11. The assay kit as claimed in claim 10, wherein either the donor oracceptor comprises lid nanoparticles which exhibit a half life of morethan 5 ns.
 12. The assay kit as claimed in claim 11, wherein either thedonor or acceptor comprises lid nanoparticles which exhibit a half lifebetween 1 μs and 50 ms.
 13. The assay kit as claimed in claim 12,wherein either the donor or acceptor comprises lid nanoparticles exhibita half life between 100 μs and 10 ms.
 14. The assay kit as claimed inclaim 1, wherein the lid nanoparticles have a breadth in the range offrom 1 nm to 50 nm.
 15. The assay kit as claimed in claim 14, whereinthe lid nanoparticles have a breadth in the range of from 1 nm to lessthan 20 nm.
 16. The assay kit as claimed in claim 15, wherein the lidnanoparticles have a breadth in the range of from 2 nm to 10 nm.
 17. Theassay kit as claimed in claim 1, wherein the lid nanoparticles exhibit aneedle-shaped morphology having a breadth in the range from 3 nm to 50nm and a length in the range from 20 nm to 5 μm.
 18. The assay kit asclaimed in one of claim 17, wherein the lid nanoparticles exhibit aneedle-shaped morphology having a breadth in the range from 3 nm to 20nm and/or a length in the range from 20 nm to 500 nm.
 19. The assay kitas claimed in claim 1, wherein a host material of the lid nanoparticlescomprises compounds of the type XY, where X is a cation consisting ofone or more elements of main groups 1a, 2a, 3a or 4a, of the subgroups2b, 3b, 4b, 5b, 6b or 7b, or of the lanthanides, of the Periodic Table,and Y is either a polyatomic anion consisting of one or more element(s)of the main groups 3a, 4a or 5a, or of the subgroups 3b, 4b, 5b, 6b, 7band/or 8b, and also element(s) of the main groups 6a and/or 7, or elseis a monoatomic anion from the main group 5a, 6a or 7a of the PeriodicTable.
 20. The assay kit as claimed claim 1, wherein a host material ofthe lid nanoparticles comprises at least one compound selected from thegroup consisting of sulfides, selenides, sulfoselenides, oxysulfides,borates, aluminates, gallates, silicates, germanates, phosphates,halophosphates, oxides, arsenates, vanadates, niobates, tantalates,sulfates, tungstates, molybdates, alkali metal halides and otherhalides, and nitrides.
 21. The assay kit as claimed in claim 1, whereinthe lid nanoparticles comprise a doping material comprising one or moreelements selected from the group consisting of elements of the maingroups 1a or 2a, or Al, Cr, Tl, Mn, Ag, Cu, As, Nb, Nd, Ni, Ti, In, Sb,Ga, Si, Pb, Bi, Zn or Co, and/or elements of the lanthanides.
 22. Theassay kit as claimed in claim 21, wherein the doping material comprisesa combination of two or more of said elements, in different relativeconcentrations to each other.
 23. The assay kit as claimed in claim 1,wherein the lid nanoparticles comprise a doping material in aconcentration of the doping material in a host lattice of between 10⁻⁵mol % and 50 mol %.
 24. The assay kit as claimed in claim 23, whereinthe concentration of the doping material in the host lattice is between0.01 mol % and 30 mol %.
 25. The assay kit as claimed in claim 24,wherein the concentration of the doping material in the host lattice isbetween 0.1 mol % and 20 mol %.
 26. The assay kit as claimed in claim 1,wherein the lid nanoparticles comprise a material selected from thegroup consisting of YVO₄:Eu, YVO₄:Sm, YVO₄:Dy, LaPO₄:Eu, LaPO₄:Ce,LaPO₄:Ce,Tb, LaPO₄:Ce,Dy, LaPO₄:Ce,Nd, ZnS:Th, ZnS:TbF₃, ZnS:EuZnS:EuF₃, Y₂O₃:Eu, Y₂O₂S:Eu, Y₂SiO₅:Eu, SiO₂:Dy, SiO₂:Al, Y₂O₃:Tb,CdS:Mn, ZnS:Tb, ZnS:Ag and ZnS:Cu.
 27. The assay kit as claimed in claim1, wherein the lid nanoparticles comprise a material in which the hostlattice has a cubic structure.
 28. The assay kit as claimed in claim 1,wherein the lid nanoparticles comprise a material selected from thegroup consisting of MgF₂:Mn, ZnS:Mn, ZnS:Ag, ZnS:Cu, CaSiO₃:Ln, CaS:Ln,CaO:Ln, ZnS:Ln, Y₂O₃:Ln, and MgF₂:Ln (Ln=lanthanides).
 29. The assay kitas claimed in claim 1, wherein the donor and/or the acceptor compriselid nanoparticles which, after energetic excitation with electromagneticradiation with wavelengths in the range of infrared light, of visiblelight, of UV, of X-ray light or of γ-radiation, or particle radiationemit electromagnetic radiation with a Stokes shift or anti-Stokes shift.30. The assay kit as claimed in claim 1, wherein the donor and/or theacceptor comprise lid nanoparticles which, after energetic excitationwith electromagnetic radiation with wavelengths in the range of electronradiation, emit electromagnetic radiation with a Stokes shift oranti-Stokes shift.
 31. The assay kit as claimed in claim 1, wherein thedonor comprises lid nanoparticles and the acceptor comprises aconducting material.
 32. The assay kit as claimed in claim 31, whereinthe conducting material is a metal, a conducting oxide, or a conductingpolymer.
 33. The assay kit as claimed in claim 32, wherein theconducting material is a metal selected from the group consisting ofgold, silver and platinum; the conducting oxide is indium tin oxide(ITO); and the conducting polymer is present in particulate form asnanoparticles or microparticles or as a planar, optionally structured,surface.
 34. The assay kit as claimed in claim 1, which is capable ofuse in a homogeneous assay without any washing or separating steps. 35.The assay kit as claimed in claim 1, which is capable of use in ahomogeneous immunoassay detecting at least one analyte in a sample. 36.The assay kit as claimed in claim 35, wherein the at least one analyteis selected from the group consisting of at least one monoclonal orpolyclonal antibody, protein, peptide, oligonucleotide, nucleic acid,oligosaccharide, polysaccharide, hapten and low molecular weightsynthetic or natural antigen; and/or the sample comprises at least onemember selected from the group consisting of smears, sputum, organpunctate, biopsies, secretions, spinal fluid, bile, blood, lymph fluid,urine and feces.
 37. The assay kit as claimed in claim 1, wherein asurface of the lid nanoparticle(s) is prepared such that affinitymolecules can be coupled to it.
 38. The assay kit as claimed in claim37, wherein the surface of the lid nanoparticles is chemically modifiedand/or exhibits reactive groups and/or covalently or noncovalently boundconnecting molecules, with the bound connecting molecules being able,for their part, to exhibit reactive groups.
 39. The assay kit as claimedin claim 38, wherein the reactive groups are selected from the groupconsisting of amino groups, carboxylic acid groups, thiols, thioethers,disulfides, imidazoles, guanidines, hydroxyl groups, indoles, vicinaldiols, aldehydes, alpha-haloacetyl groups, N-maleimides, mercurides,aryl halides, acid anhydrides, isocyanates, isothiocyanates, sulfonylhalides, imidoesters, diazoacetates, diazonium salts, 1,2-diketones,alpha-beta-unsaturated carbonyl compounds, phosphonic acids, phosphoricacid esters, sulfonic acids and azolides, and derivatives of saidreactive groups.
 40. The assay kit as claimed in claim 39, wherein theconnecting molecules are selected from the group consisting of nucleicacid molecules, phosphonic acid derivatives, ethylene glycol, primaryalcohols, amine derivatives, polymers or copolymers, polymerizablecoupling agents, silica shells and catenate molecules having a polaritywhich is opposite to that of the surface of the lid nanoparticles. 41.The assay kit as claimed in claim 40, wherein the polymerizable couplingagents are selected from the group consisting of diacetylenes,styrenebutadienes, vinyl acetate, acrylates, acrylamides, vinyls,styrenes, silicone oxides, boron oxides, phosphorus oxides, borates,pyrroles, polypyrroles and phosphates, and also polymers of at leastsome of said polymerizable coupling agents.
 42. The assay kit as claimedin claim 37, wherein the affinity molecules are selected from the groupconsisting of proteins, peptides, oligonucleotides or other nucleic acidmolecules or nucleic acid-like molecules, oligosaccharides orpolysaccharides, haptens, and low molecular weight synthetic naturalantigens or epitopes.
 43. The assay kit as claimed in claim 42, whereinthe nucleic acid-like molecules are PNAs or morpholinos; and/or thehaptens are biotin or digoxin.
 44. The assay kit as claimed in claim 37,wherein the affinity molecules are able to interact with targetmolecules.
 45. The assay kit as claimed in claim 44, wherein the targetmolecule is an enzyme, an antibody, a nucleic acid-binding molecule, anucleic acid, a polynucleotide or a morpholino.
 46. The assay kit asclaimed in claim 45, wherein the enzyme is endonuclease, protease,kinase or phosphatase or an amino acid- or nucleic acid-modifying orcleaving enzyme.
 47. The assay kit as claimed in claim 46, wherein aninteraction of the affinity molecule with the target molecule results ina change in a spatial separation of molecule groups A and B.
 48. Theassay kit as claimed in claim 1, wherein the molecule groups A and B areconstituents of one and the same molecule.
 49. The assay kit as claimedin claim 1, wherein the molecule groups A and B are able to couple tothe same affinity molecule.
 50. The assay kit as claimed in claim 1,which is used for quantifying nucleic acids.
 51. The assay kit asclaimed in claim 1, wherein the molecule groups A and B are constituentsof different molecules.
 52. The assay kit as claimed in claim 51,wherein the molecule groups A and B are in each case coupled to theirown affinity molecules.
 53. The assay kit as claimed in claim 52,wherein the affinity molecules which are assigned to molecule groups Aand B are able to interact specifically with the same target molecule.54. The assay kit as claimed in claim 53, wherein an interaction of theaffinity molecules which are assigned to molecule groups A and B withthe common target molecule or with each other result in a change in thespatial separation of molecule groups A and B.
 55. The assay kit asclaimed in claim 52, wherein the affinity molecules which are assignedto molecule groups A and B are able to interact specifically with eachother.
 56. The assay kit as claimed in claim 1, wherein the lidnanoparticles comprise a material selected from the group consisting ofLiI:Eu; NaI:Tl; CsI:Tl; CsI:Na; Lif:Mg; LiF:Mg,Ti; LiF:Mg,Na; KMgF₃:Mn;Al₂O₃:Eu; BaFCl:Eu; BaFCl:Sm; BaFBr:Eu; BaFCl_(0.5)Br_(0.5):Sm; BaY₂F₈:A(A=Pr, Tm, Er or Ce); BaSi₂O₅:Pb; BaMg₂Al₁₆O₂₇:Eu; BaMgAl₁₄O₂₃:Eu;BaMgAl₁₀O₁₇:Eu; BaMgAl₂O₃:Eu; Ba₂P₂O₇:Ti; (Ba,Zn or Mg)₃Si₂O₇:Pb; Ce(Mgor Ba)Al₁₁O₁₉; Ce_(0.65) Tb_(0.35)MgAl₁₁O₁₉:(Ce or Tb); MgAl₁₁O₁₉:(Ce orTb); MgF₂:Mn; MgS:Eu; MgS:Ce; MgS:Sm; MgS:(Sm or Ce); (Mg or Ca)S:Eu;MgSiO₃:Mn; 3.5MgO.0.5MgF₂.GeO₂:Mn; MgWO₄: Sm; MgWO₄:Pb, 6MgO.As₂O₅:Mn;(Zn or Mg)F₂:Mn; (Zn₄Be)SO₄:Mn; Zn₂SiO₄:Mn; Zn₂SiO₄:Mn,As; ZnO:Zn;ZnO:Zn, Si,Ga; Zn₃(PO₄)₂Mn; ZnS:A (A=Ag, Al or Cu); (Zn or Cd)S:A (A=Cu,Al, Ag or Ni); CdBO₄:Mn; CaF₂:Mn; CaF₂:Dy; CaS:A (A=lanthanides or Bi);(Ca or Sr)S:Bi; CaWO₄:Pb; CaWO₄:Sm; CaSO₄:A (A=Mn or lanthanides);3Ca₃(PO₄)₂.Ca(F or Cl)₂:Sb,M_(n); CaSiO₃:(Mn or Pb); Ca₂Al₂Si₂O₇:Ce; (Caor Mg)SiO₃:Ce; (Ca or Mg)SiO₃:Ti; 2SrO.6(B₂O₃).SrF₂:Eu;3Sr₃(PO₄)₂.CaCl₂:Eu; A₃(PO₄)₂.ACl₂:Eu (A=Sr, Ca or Ba); (Sr orMg)₂P₂O₇:Eu; (Sr or Mg)₃(PO₄)₂:Sn; SrS:Ce; SrS:Sm,Ce; SrS:Sm; SrS:Eu;SrS:Eu,Sm; SrS:(Cu or Ag); Sr₂P₂O₇:Sn; Sr₂P₂O₇:Eu; Sr₄Al₁₄O₂₅:Eu;SrGa₂S₄A (A=lanthanides or Pb); SrGa₂S₄:Pb; Sr₃Gd₂Si₆O₁₈:Pb,Mn;YF₃:Yb,Er; YF₃:Ln (Ln=lanthanides); YLiF₄:Ln (Ln=lanthanides);Y₃Al₅O12:Ln (Lu=lanthanides); YAl₃(BO₄)₃:(Nd or Yb); (Y or Ga)BO₃:Eu; (Yor Gd)BO₃:Eu; Y₂Al₃Ga₂O₁₂:Tb; Y₂SiO₅:Ln (Ln=lanthanides); Y₂O₃:Ln(Ln=lanthanides); Y₂O₂S:Ln (Lu=lanthanides); YVO₄:A (A=lanthanides orIn); Y(P,V)O₄:Eu; YTaO₄:Nb; YAlO₃:A (A=Pr, Tm, Er or Ce); YOCl:(Yb orEr); LnPO₄: (LnCe or Tb=lanthanides or mixtures of lanthanides);LuVO₄:Eu; GdVO₄:Eu; Gd₂O₂S:Tb; GdMgB₅O₁₀:(Ce or Tb); LaOBr:Tb;La₂O₂S:Tb; LaF₃(Nd or Ce); BaYb₂F₈:Eu; NaYF₄:(Yb or Er); NaGdF₄:(Yb orEr); NaLaF₄:(Yb or Er); LaF₃:Yb, Er or Tm); BaYF₅:(Yb or Er); Ga₂O₃:Dy;GaN:A (A=Pr, Eu, Er or Tm); Bi₄Ge₃O₁₂; LiNbO₃:(Nd or Yb); LiNbO₃:Er;LiCaAlF₆:Ce; LiSrAlF₆:Ce; LiLuF₄:A (A=Pr, Tm, Er or Ce); Li₂B₄O₇:Mn, andSiO_(x):(Er or Al) (O≦x≦2).
 57. A method for detecting a targetmolecule, comprising the steps of: a) providing an assay kit as claimedin claim 1,  wherein, in the assay kit, the molecule groups A and B areconstituents of one and the same molecule and couple to the sameaffinity molecule, which is capable of interacting with a specifictarget molecule, and such an interaction brings about a change in theseparation between the molecule groups A and B, b) adding a samplecontaining the target molecule to the assay kit, c) exciting the assaykit containing the sample with a source of electromagnetic orparticulate radiation, and d) measuring the electromagnetic radiationemitted by the assay kit containing the sample,  wherein the intensityor the spectrum of the emitted electromagnetic radiation, or thechronological course of the emission of the electromagnetic radiation,is a measure of the quantity of target molecule in the sample.
 58. Amethod for detecting a target molecule, comprising the steps of: a)providing an assay kit as claimed in claim 1,  wherein the moleculegroups A and B are constituents of different molecules, and  theaffinity molecules which are assigned to molecule groups A and B arecapable of specifically interacting with one and the same targetmolecule, or  the affinity molecules which are assigned to moleculegroups A and B are capable of specifically interacting with each other, and in both cases an interaction brings about a change in theseparation between molecule groups A and B, b) adding a samplecontaining the target molecule to the assay kit, c) exciting the assaykit containing the sample with a source of electromagnetic orparticulate radiation, and d) measuring the electromagnetic radiationemitted by the assay kit containing the sample,  where the intensity orthe spectrum of the emitted electromagnetic radiation, or thechronological course of the emission of the electromagnetic radiation,is a measure of the quantity of target molecule in the sample.